In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

Kinetic Parameters of the Conversion of Methane Precursors to Methane in a Hypereutrophic Lake Sediment

The kinetic parameters K_m, V_max, T_t (turnover time), and v (natural velocity) were determined for H₂ and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14°C. Estimates of the Michaelis-Menten constant, K_m, for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 μmol g⁻¹ of dry sediment. The maximal velocity, V_max, averaged 4.8 μmol of H₂ g⁻¹ h⁻¹ for hydrogen consumption and 0.64 μmol of CH₄ g⁻¹ h⁻¹ for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K_m, V_max, and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-¹⁴C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH₄ g⁻¹ h⁻¹, which was 66% of the V_max (163 nmol of CH₄ g⁻¹ h⁻¹). A 15-min preincubation of sediment with 5.0 × 10⁻³ atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K_m + S_n (S_n is in situ substrate concentration), decreased by 37% and T_t decreased by 43%. The V_max remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-¹⁴C]valine by sediment heterotrophs.     

Prokaryotic Community Structure Driven by Salinity and Ionic Concentrations in Plateau Lakes of the Tibetan Plateau

The prokaryotic community composition and diversity and the distribution patterns at various taxonomic levels across gradients of salinity and physiochemical properties in the surface waters of seven plateau lakes in the Qaidam Basin, Tibetan Plateau, were evaluated using Illumina MiSeq sequencing. These lakes included Lakes Keluke (salinity, <1 g/liter), Qing (salinity, 5.5 to 6.6 g/liter), Tuosu (salinity, 24 to 35 g/liter), Dasugan (salinity, 30 to 33 g/liter), Gahai (salinity, 92 to 96 g/liter), Xiaochaidan (salinity, 94 to 99 g/liter), and Gasikule (salinity, 317 to 344 g/liter). The communities were dominated by Bacteria in lakes with salinities of <100 g/liter and by Archaea in Lake Gasikule. The clades At12OctB3 and Salinibacter, previously reported only in hypersaline environments, were found in a hyposaline lake (salinity, 5.5 to 6.6 g/liter) at an abundance of ∼1.0%, indicating their ecological plasticity. Salinity and the concentrations of the chemical ions whose concentrations covary with salinity (Mg²⁺, K⁺, Cl⁻, Na⁺, SO₄²⁻, and Ca²⁺) were found to be the primary environmental factors that directly or indirectly determined the composition and diversity at the level of individual clades as well as entire prokaryotic communities. The distribution patterns of two phyla, five classes, five orders, five families, and three genera were well predicted by salinity. The variation of the prokaryotic community structure also significantly correlated with the dissolved oxygen concentration, pH, the total nitrogen concentration, and the PO₄³⁻ concentration. Such correlations varied depending on the taxonomic level, demonstrating the importance of comprehensive correlation analyses at various taxonomic levels in evaluating the effects of environmental variable factors on prokaryotic community structures. Our findings clarify the distribution patterns of the prokaryotic community composition in plateau lakes at the levels of individual clades as well as whole communities along gradients of salinity and ionic concentrations.     

Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture

Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ_max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h⁻¹), specific maintenance rate (0.11 h⁻¹), actual molar growth yield at μ_max (Y_max = 16 g mol⁻¹), and theoretical molar growth yield (Y_G = 24 g mol⁻¹) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h⁻¹) and lowest at 70 and 75°C (0.11 and 0.15 h⁻¹, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ_max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements.     

The Impact of Gamma Radiation on Sediment Microbial Processes

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h⁻¹ for 8 weeks all displayed NO₃⁻ and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h⁻¹ treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h⁻¹ treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO₃⁻, Fe(III), or SO₄²⁻ reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h⁻¹-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h⁻¹ and 30-Gy h⁻¹ treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Synergistic Interaction Between Anabaena and Zoogloea spp. in Carbon Dioxide-Limited Continuous Cultures

Flocs consisting of Anabaena and Zoogloea spp. were used as a model system for the study of planktonic phototroph-heterotroph interactions. In CO₂-limited continuous culture (3.2 μmol of NaHCO₃ liter⁻¹ h⁻¹, 1.5 μmol of glucose liter⁻¹ h⁻¹, pH 8.5, D = 0.026 h⁻¹), the biomass of the phototroph increased 8.6-fold due to association. However, direct CO₂ exchange accounted for only a 3.8-fold increase. When the glucose supply rate was increased to 7.5 μmol liter⁻¹ h⁻¹, there was a 26-fold increase in biomass. When CO₂ was supplied in excess, there was no difference due to association. In batch culture, using the same medium, the specific growth rate was 0.029 h⁻¹ for the phototroph alone and 0.047 h⁻¹ for the phototroph in association with the heterotroph. The stimulatory effect of the heterotroph was found only under CO₂-limiting conditions and was directly related to the concentration of organic matter supplied in the medium. Both the biomass and the growth rate of the Anabaena sp. were increased by association with the Zoogloea sp. Thus, dissolved organic matter may substitute for CO₂ to maximize both growth rate and biomass production by phototrophs when heterotrophic bacteria are present.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations

Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO₃⁻, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO₃⁻in vivo, −NO₃⁻in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO₃⁻ to the in vivo NR assay medium increased activity (over that of the −NO₃⁻in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO₃⁻ was rate-limiting. The stimulation of in vivo NR activity by NO₃⁻ was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO₃⁻ was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO₂⁻ [g fresh weight, hr]⁻¹) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.     

Malonic Semialdehyde Reductase from the Archaeon Nitrosopumilus maritimus Is Involved in the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

The recently described ammonia-oxidizing archaea of the phylum Thaumarchaeota are highly abundant in marine, geothermal, and terrestrial environments. All characterized representatives of this phylum are aerobic chemolithoautotrophic ammonia oxidizers assimilating inorganic carbon via a recently described thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Although some genes coding for the enzymes of this cycle have been identified in the genomes of Thaumarchaeota, many other genes of the cycle are not homologous to the characterized enzymes from other species and can therefore not be identified bioinformatically. Here we report the identification and characterization of malonic semialdehyde reductase Nmar_1110 in the cultured marine thaumarchaeon Nitrosopumilus maritimus. This enzyme, which catalyzes the reduction of malonic semialdehyde with NAD(P)H to 3-hydroxypropionate, belongs to the family of iron-containing alcohol dehydrogenases and is not homologous to malonic semialdehyde reductases from Chloroflexus aurantiacus and Metallosphaera sedula. It is highly specific to malonic semialdehyde (K_m, 0.11 mM; V_max, 86.9 μmol min⁻¹ mg⁻¹ of protein) and exhibits only low activity with succinic semialdehyde (K_m, 4.26 mM; V_max, 18.5 μmol min⁻¹ mg⁻¹ of protein). Homologues of N. maritimus malonic semialdehyde reductase can be found in the genomes of all Thaumarchaeota sequenced so far and form a well-defined cluster in the phylogenetic tree of iron-containing alcohol dehydrogenases. We conclude that malonic semialdehyde reductase can be regarded as a characteristic enzyme for the thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle.     

Estimation of Sediment Denitrification Rates at In Situ Nitrate Concentrations

The denitrification rates in a marine sediment, estimated by using ¹⁵N-nitrate, V_max, K_m, and sediment nitrate concentrations, were 12.5 and 2.0 nmol of N₂-N cm⁻³ day⁻¹ at 0 to 1 and 1 to 3 cm, respectively, at 12°C. The total rate was 165 nmol of N₂-N m⁻² day⁻¹.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Comparison of Extracellular Enzyme Activities and Community Composition of Attached and Free-Living Bacteria in Porous Medium Columns

Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10⁻⁷ nmol cell⁻¹ h⁻¹) than the free-living community (5.02 × 10⁻⁸ nmol cell⁻¹ h⁻¹). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10⁻⁶ nmol cell⁻¹ h⁻¹) than the surface-associated community (6.08 × 10⁻⁷ nmol cell⁻¹ h⁻¹). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.     

Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Pyrophosphorylases in Solanum tuberosum: II. CATALYTIC PROPERTIES AND REGULATION OF ADP-GLUCOSE AND UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITIES IN POTATOES

Pyrophosphorylytic kinetic constants (S_0.5, V_max) of partially purified UDP-glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S_0.5 of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the V_max were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A_0.5 = 4.5 × 10⁻⁶ molar). The S_0.5 for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P-glycerate while the V_max was increased nearly 4-fold. The magnitude of 3-P-glycerate stimulation was dependent upon the integrity of key sulfhydryl groups (−SH) and pH. Oxidation or blockage of −SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I_0.5 = 8.8 × 10⁻⁵. molar). This inhibition was reversed with 3-P-glycerate (1.2 × 10⁻⁴ molar), resulting in an increased I_0.5 value of 1.5 × 10⁻³ molar. Likewise, orthophosphate (7.5 × 10⁻⁴ molar) caused a decrease in the activation efficiency of 3-P-glycerate (A_0.5 from 4.5 × 10⁻⁶ molar to 6.7 × 10⁻⁵ molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of α-glucan biosynthesis in Solanum tuberosum is discussed.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Gammaproteobacterial Methanotrophs Dominate Cold Methane Seeps in Floodplains of West Siberian Rivers

A complex system of muddy fluid-discharging and methane (CH₄)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH₄ flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH₄ h⁻¹, while some seeps emitted up to 5.54 g CH₄ h⁻¹. The δ¹³C value of methane released from these seeps varied between −71.1 and −71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH₄ ml⁻¹ day⁻¹) were measured in mud samples. Fluorescence in situ hybridization detected 10⁷ methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH₄ sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.     

Hydrogen Metabolism by Decomposing Cyanobacterial Aggregates in Big Soda Lake, Nevada

Hydrogen production by incubated cyanobacterial epiphytes occurred only in the dark, was stimulated by C₂H₂, and was inhibited by O₂. Addition of NO₃⁻ inhibited dark, anaerobic H₂ production, whereas the addition of NH₄⁺ inhibited N₂ fixation (C₂H₂ reduction) but not dark H₂ production. Aerobically incubated cyanobacterial aggregates consumed H₂, but light-incubated rates (3.6 μmol of H₂ g⁻¹ h⁻¹) were statistically equivalent to dark uptake rates (4.8 μmol of H₂ g⁻¹ h⁻¹), which were statistically equivalent to dark, anaerobic production rates (2.5 to 10 μmol of H₂ g⁻¹ h⁻¹). Production rates of H₂ were fourfold higher for aggregates in a more advanced stage of decomposition. Enrichment cultures of H₂-producing fermentative bacteria were recovered from freshly harvested, H₂-producing cyanobacterial aggregates. Hydrogen production in these cyanobacterial communities appears to be caused by the resident bacterial flora and not by the cyanobacteria. In situ areal estimates of dark H₂ production by submerged epiphytes (6.8 μmol of H₂ m⁻² h⁻¹) were much lower than rates of light-driven N₂ fixation by the epiphytic cyanobacteria (310 μmol of C₂H₄ m⁻² h⁻¹).